CHAPTER - IV - Shodhganga : a reservoir of Indian theses ...shodhganga.inflibnet.ac.in/bitstream/10603/4370/11/11...chocolate syrup, bread-making, fruit juices, paper and desizing

Enzyme Profiling of Actinomycetal Isolates

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CHAPTER - IV

ENZYME PROFILING OF ACTINOMYCETAL ISOLATES

4.1 INTRODUCTION

This chapter deals with the screening of indigenously isolated Actinomycetes for

glucose isomerase production. The basic idea behind enzyme profiling is to use these

isolates for application of glucose isomerase. Production of High Fructose Corn Syrup

(HFCS) requires glucose isomerase and the raw materials used for this are cellulosic

or starchy, therefore an organism possessing multiple enzyme producing capability

will be of great industrial importance. The production of HFCS is done by using non

sweet polysaccharide materials. The economically available agro-residues are rich in

starch, cellulose and protein material. These enzymes are also useful in degrading

above substrates for ethanol production. Various researchers have prepared a

cocktail of polysaccharide degrading enzyme with isomerising enzyme and then

subjecting it to fermentation by yeasts. As such the Streptomycetes are known to

produce a large number of enzymes; we have screened the possibility of using our

isolates for degrading the macromolecular raw materials for HFCS production.

A wide variety of bacteria are known for their production of hydrolytic enzymes

with Streptomycetes being the best known enzyme producers. They are capable of

secreting an array of different extracellular enzymes including glucose isomerase,

amylase, cellulase, lipase, protease, pectinase, keratinase, L-asparginase, chitinases,

and xylanases. Streptomycetes are known to produce a variety of industrially

important enzymes.

Production of glucose isomerase is widely done from Streptomycetes which has

already been dealt in Chapter – I in detail.

Amylases have most widely been reported to occur in microorganisms.

Streptomyces species, the saprophytic microorganisms use these enzymes to degrade

polymeric nutrients in soil originating from decaying plant material. Several genes

encoding extracellular enzymes have been cloned from Streptomyces species. These


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include endoglucosidase, tyrosinase, agarase, lactamase, amylase, and xylanase. In

addition to well established applications in starch saccharification and in the textile,

food, brewing, distilling industries, preparation of pharmaceuticals, chemicals,

chocolate syrup, bread-making, fruit juices, paper and desizing of textiles bacterial

amylases are now also used in areas of clinical, medicinal, and analytical chemistry.

There are two main requirements in the production process of sweeteners from

starch: the temperature should be 50°C or more and pH close to 7 to prevent the

browning effects and to reduce the viscosity of starch pastes.

Thermostable α-amylases are generally preferred as their application

minimizes contamination risk and reduces reaction time, thus providing considerable

energy saving. Hydrolysis carried out at higher temperature also minimizes

polymerization of D-glucose to isomaltose [Pandey et al., 2005].

Amylase is produced at industrial level by Bacillus sp. and fungus mainly

Aspergillus sp. Most of the amylases are inducible and are produced in presence of

starch. Organic nitrogen sources like peptone, tryptone and yeast extract are mainly

preferred for the production of amylase. Suitable pH for production of amylase falls

between 6 and 7 for majority of the reported isolates. Alkaline amylase producing

organisms have great market applications [Aiyer and Modi, 2005]. Temperature

range for amylase varies with the type of isolates [Kar et al., 2011; Gulve and

Deshmukh, 2011; Khosravi-Darani et al., 2008; Kar et al., 2008; Sharma and

Shukla, 2007; Kurosawa et al., 2006; Ajayi et al., 2006]. Various strain

improvement strategies have been opted by researchers for the production of amylase

like mutagenesis, genetic engineering, modulation of ribosomal protein etc.

[Kurosawa et al., 2006; Garcia-Gonzalez et al., 1991].

Cellulose is the most common organic polymer, representing about 1.5x 1012

tonnes of the total annual biomass production through photosynthesis especially in

the tropics, and is considered to be an almost inexhaustible source of raw material

for different products. It is the most abundant and renewable biopolymer on earth

and the dominating waste material from agriculture. Lignocellulose, the structural

material of plant cell wall contains about 30 to 60% of cellulose. A promising strategy

for efficient utilization of this renewable resource is the microbial hydrolysis of


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lignocellulosic waste and fermentation of the resultant reducing sugars for

production of desired metabolites or biofuel. Potent cellulolytic organisms produce a

complex mixture of enzymes required for the efficient solublisation of the substrate

[Acharya et al. 2010; Sukumaran et al., 2005; Jang and Chang, 2005, Modi et

al., 1994].

Samedo et al., (2000) isolated Streptomyces sp. from forest soil which

produced seven times higher cellulase than Trichoderma reesie. Cappa et al., (1997)

cloned the endoglucanase gene from Streptomyces rochei, a cellulolytic and

ligninolytic bacteria living in endosymbiontic association with termites into strains of

Ruminococcus albus.

Cellulase is an industrially important enzyme, which is extensively used for

increasing yield of juice in food industry, decreasing discoloration and fuzzing effects

of cloth in textile industry, strengthening and whitening of paper pulp in paper

industry, and to biofuel generation through saccharification process. Actinomycetes,

one of the known cellulase-producers, has attracted considerable research interest

due to its potential applications in recovery of fermentable sugars from cellulose that

can be of benefit for human consumption and to the ease of their growth. Cellulose

decomposing bacteria and fungi, widely distributed in the environment, play an

important role as mineralizers of organic matter and thereby influencing productivity.

Cellulose from plant biomass is the only foreseeable sustainable source of fuels and

materials available to humanity. Cellulose materials are particularly attractive in this

context because of their relatively low cost and plentiful supply [Jaradat et al.,

2008; Nurkanto, 2009; Murugan et al., 2007].

Actinomycetes, particularly Streptomycetes are known to secrete multiple

proteases in culture medium. Proteases may conveniently be produced by

fermentation using cheap substrate such as wheat bran. Protease is an industrially

important enzyme having wider applications in pharmaceutical, leather, laundry,

detergent, i.e., help in removing protein based stains from clothing, leather

preparation, meat tenderization, peptide synthesis , food industry, dehairing process,

pharmaceutical industry (in contact lens eye cleaners) as well as in bioremediation

process. Industrial enzyme production would be effective only if the organism and the

target enzyme are capable of tolerating different variables of the production


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processes. Proteases are among the most important class of industrial enzymes,

which constitute more than 65% of the total sales of industrial enzymes and around

500 tonnes of protease enzyme are produced every year to fulfill demand coming

from industries [Crueger and Crueger, 1984]. Two-thirds of the proteases produced

commercially are of microbial origin [Guravaiah et al., 2010; Ningthoujam et al.,

2009; Guangrong et al., 2008; Khosravi-Darani et al., 2008; Mehta et al., 2006;

Rifaat et al., 2006].

Yang and Wang, (1999) compared the submerged and solid state fermentative

production process for protease and amylase from Streptomyces rimosus. Amylase

and protease production reached maximum in 48 h and 166 h respectively for S.

rimosus. Amylase activity was connected with the substrate utilization by microbes

while protease was associated with the growth of microbes. Therefore, amylase was

secreted prior to protease. Jang and Chang, (2005) reported the production of

thermostable cellulases from Streptomyces sp. in a 50 liter fermentor.

Vonothini et al., (2008) isolated Streptomyces sp. from esturine shrimp pond

which exhibited high protease production in the presence of sucrose, L-glutamine

and 3% sodium chloride at pH 7 and temperature 40°C. Kathiresan and

Manivannan, (2007) isolated Streptomyces sp. which produced alkaline protease in

a medium containing 5% sucrose and 7.5% gelatin in 120 h at 30°C and pH 8.5.

Production of protease is greatly influenced by nitrogen source present in the

medium [Bascarn et al., 1990].

The collection of 75 isolates developed was used to perform enzyme profiling

studies. The isolates which are selected on the basis of qualitative plate assay

method were checked for GI production by submerged fermentation process. The

qualitative method revealed 36 out of 75 isolates probably producing glucose

isomerases which were further taken up for following analysis.

The screening strategy which we designed for glucose isomerase producers

was in the absence of xylose, the inducer of the enzyme. Xylose is usually

incorporated in the medium for effective screening method. The idea behind this was

to select isolates which can produce GI in detectable amounts without adding an

inducer. An isolate which can produce GI in absence of pure xylose will definitely


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produce higher amounts in it’s presence. Such an organism may also produce higher

amounts of the enzyme in the presence of crude sources of xylose. Wheat bran,

peanut shell and hemicelluloses are rich in xylan as well as xylose content.

4.2 MATERIALS AND METHODS

The screening process was started with plate assay method to check the

production of enzymes qualitatively. The isolates giving considerably large zone

diameters were further picked up for producing the enzymes by submerged

fermentation as a secondary screening process.

4.2.1 Qualitative Analysis

Qualitative analysis for the production of different enzymes was done by

incorporating their respective substrates in agar medium for plate assay method.

4.2.1.1 Glucose Isomerase production

The purified isolates were screened for production of enzyme by using specific

substrate containing media. Production of glucose isomerase was checked on media

containing xylose as a sole source of carbon and wheat bran media. The organisms

producing glucose isomerase can isomerise xylose to xylulose besides glucose to

fructose. The organisms growing on medium containing xylose as a sole source of

carbon will be utilizing xylose as a source of carbon. Xylose has to be first converted

to xylulose which is further channelized into pentose phosphate pathway for

generation of energy. The organisms possessing very low or negligible GI activity

might not grow on such a media. The screening strategy was designed according to

the method described by Manhas and Bala, (2004) with some modifications. We

used three different media combinations for primary screening, X+P+ medium, X+P-

medium and wheat bran medium as described in appendix I. The cultures were spot

inoculated on all the media combinations and incubated at 30°C. The plates were

observed daily. The isolates developing early on the plates and giving luxurious

growth were picked up as GI producers.


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4.2.1.2 Amylase Production

Screening for amylase was done on Bennett’s agar medium containing 1% starch

(Appendix - I). The purified isolates were spot inoculated in the centre of the

petridish. The plates were incubated at 30°C. The plates were observed daily and the

zone of hydrolysis noted on the fifth day of incubation. The plates were flooded with

Lugol’s iodine solution (Appendix - II). The area near the organism’s growth remained

colourless and rest of the media in the plate took blue colour indicating the presence

of starch [Kar et al., 2008].

4.2.1.3 Cellulase production

Bennett’s agar medium containing 1% cellulose was used for checking cellulase

production (Appendix - I). The purified cultures were spot inoculated in the centre of

the petridish and incubated at 30°C. The plates were observed daily and the zone of

hydrolysis noted on the fifth day of incubation. Lugol’s iodine solution (Appendix - II)

was poured on the plates to observe the zone of hydrolysis. The clear area around the

zone indicated the cellulase production and media in rest of the plates gave reddish

brown colour [Kasana et. al., 2008].

4.2.1.4 Protease production

Protease production was observed on plates containing different protein

sources. Production of gelatinase and caseinase enzyme was checked. Gelatin agar

plates were prepared by adding gelatin to Bennett’s agar and Casein agar plates were

prepared by adding casein to Bennett’s agar (Appendix - I). The purified isolates were

inoculated in the centre of the plates. Incubation was done at 30°C. The plates were

observed daily. Casein hydrolysis was observed by development of a cream coloured

halo around the colonies which kept on increasing with time. Gelatinase production

was observed by adding commassie brilliant blue (Appendix - II) to the plates and

allowing it to develop for 1h. The area around the gelatinase producing organisms

was lighter than rest of the plate [Vermelho et al., 1996].


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4.2.1.5 Lipase production

Production of lipase by the Actinomycete isolates was checked on Tri butyrene

agar plates (Appendix - I). The cultures were spot inoculated in the centre of the

plates and incubated at 30°C. The plates were observed daily and the lipolytic activity

could be observed by clear zones around the colonies. The zone diameters were

measured on the fifth day.

4.2.1.6 Pectinase production

Pectinase production was checked by incorporating 1% pectin in the Bennett’s

agar media (Appendix - I). The purified isolates were spot inoculated in the centre of

the plate and incubated at 30°C. The zone of hydrolysis was checked by flooding the

plates with 1% alcoholic CTAB (cetyl tri-methyl ammonium bromide) (Appendix - II).

The cultures having pectinase activity developed clear zones around them after an

incubation period of 1 hour [Saadoun et al., 2007; Kobayashi et al., 1999].

4.2.2 Quantitative Analysis

The isolates which were found to be good producers at qualitative analysis

level were picked up for further studies. They were subjected to submerged

fermentation in liquid media to check the amount of extracellular enzyme produced.

Conical flasks containing 20 mL of Bennett’s broth (Appendix - I) were sterilized by

autoclaving. The selected isolates were inoculated in Bennett’s broth of pH 7 and

incubated in orbital shaker. The fermentation was terminated on the fourth day and

broth was harvested in sterile centrifuge tubes. The fermented broth was centrifuged

at 5000 RPM for 10 minutes. The supernatant was used as crude extracellular

enzyme extract. Quantitative analysis was performed for amylase, cellulase, protease

and glucose isomerase.

4.2.2.1 Screening for Glucose isomerase production by submerged fermentation

The centrifuged supernatant of fermented broth was used as crude enzyme extract

for determination of GI activity by assay method described by Chen et al, (1979).

The reaction mixture for GI assay contained 500 µL of 0.2 Molar sodium phosphate

buffer, 200 µL of 1 M glucose, 100 µL of 0.1 M magnesium sulphate, 100 µL of 0.01


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M cobalt chloride and 200 µL of crude enzyme extract. The final volume of assay

mixture was made up to 2 mL. This reaction mixture was incubated in water bath at

70°C for 60 minutes. The reaction was stopped by adding 2 mL of 0.5 M perchloric

acid. To 0.05 mL aliquot of above 0.95 mL of distilled water was added. To this 200

µL of 1.5% cysteine hydrochloride, 6mL of 70% sulphuric acid and 200 µL of 0.12%

alcoholic Carbazole is added. The intensity of purple colour so developed was

estimated spectrophotometrically at 560 nm [Dische and Borenfreund, 1951]. One

unit of glucose isomerase activity was defined as the amount of enzyme that

produced 1μmol of fructose per minute under the assay conditions described.

4.2.2.2 Screening for Amylase production by submerged fermentation

The cultures producing large hydrolysis zones on Starch agar plates were

picked up for amylase production by submerged fermentation. The production and

enzyme extract preparation was done according to the method described in section

4.2.2. Amylase assay was performed by estimation of reducing sugar produced from

1% starch solution by Dinitrosalicylic acid method [Miller, 1959]. Amylase was

assayed by adding 0.5ml of 1% soluble starch solution to 0.2ml of enzyme extract.

The reaction mixture was incubated at 37°C in the waterbath for 30 mins. The

reaction was stopped by adding 1ml DNS reagent. The volume was made up to 5 ml.

and absorbance was measured at 540nm. One unit of enzymatic activity was defined

as the amount of enzyme required to produce 1 µM/minute of glucose under the

assay conditions [Manivasagan et al., 2010].

4.2.2.3 Screening for Cellulase production by submerged fermentation

Isolates selected by plate assay method were subjected to submerged

fermentation. The production of enzyme and enzyme extract preparation was done

according to the details given in section 4.2.2. Filter paper activity for cellulase was

determined according to method proposed by Semedo et al., (2000) with some

modifications. The activity was determined by adding 0.5ml of culture filtrate to

0.5ml of 0.05M Citrate buffer and 50mg. filter paper. The reaction mixture was

incubated at 50°C for 1 hour. The reaction was stopped by adding 1ml DNS reagent.

The volume was made up to 12ml. and absorbance was measured at 540nm. One


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unit of cellulase was defined as the amount of enzyme which produced 1 μ mole

glucose equivalent per minute under the assay conditions.

4.2.2.4 Screening for Protease production by submerged fermentation

The isolates producing large zones on milk, gelatin and casein agar plates

were picked up as good protease producers to perform secondary screening. The

production of enzyme and enzyme extract preparation was done according to the

details given in section 4.2.2. Protease assay is a modification of two methods viz.

Hagihara, 1953 and Anson, 1938. Casein was used as the substrate and protease

activity was determined by estimating the soluble tyrosine released. Tyrosine was

determined colorimetrically using Folin reagent. To 5ml of purified casein solution

1ml of crude enzyme extract (1:1 diluted with acetate buffer) was added. This was

incubated for 10 minutes at 30°C. The reaction was terminated by adding 5ml of tri-

chloro acetic acid. This was incubated at 30°C for 30 minutes for precipitating

remaining total protein. The precipitates were separated by centrifugation. 2 ml of

supernatant was mixed with 5 ml of sodium carbonate and 1ml of 1N Folin reagent.

This was incubated for 30 minutes at 30°C. The absorbance for determination of

tyrosine value (γ) was measured at 660 nm and expressed in Proteolytic Unit of

Nagase (PUN). One PUN is defined as the amount enzyme which acts on casein for 10

minutes at 30°C and produces a quantity of Folin color-producing substances not

precipitated by trichloroacetic acid, that is equivalent to 1 γ of tyrosine.

4.3 RESULTS AND DISSCUSSION

4.3.1 Qualitative Analysis

Streptomycetes are slow growing organisms as they have a programmed life

cycle. The spores germinate to form mycelium which branches extensively to anchor

on the agar surface. The mature substrate hyphae gives rise to aerial hyphae which

further develop spores. Sporulation usually takes three to four days. As the growth of

the organism itself is slow therefore the enzyme production was observed after

maturing of the colonies. The hydrolysis zones were measured on fifth day of

incubation for all enzymes. Enzyme profile of all 75 isolates is shown in Table 4.1.


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Table No. 4.1: Enzyme profile of 75 isolates.

Sr. No. Isolate

Code

Glucose

Isomerase

Cellulase

(m.m.)

Amylase

(m.m.)

Lipase

(m.m.)

Caseinase

(m.m.)

Gelatinase

(m.m.)

Pectinase

(m.m.)

1. P1 ++++ 40 30 20 ND ND 32

2. P2 ++ 37 35 ND 22 ND 40

3. V1 ++ 58 41 ND ND ND 26

4. V2 - 22 26 26 ND ND ND

5. V3 ++ 25 27 25 44 22 ND

6. V4 - 45 45 26 40 42 22

7. V5 ++++ 44 21 47 ND ND 28

8. V6 ++ 41 48 38 ND ND ND

9. V7 ++ 44 35 29 ND ND 37

10. Ab ++ 34 55 39 65 65 ND

11. Ga1 - 68 45 26 46 65 ND

12. Ga2 - ND ND 22 40 ND 13

13. Ga3 - 19 ND 40 47 30 15

14. Ga4 - 19 ND 50 40 ND 15

15. Gu1 - 22 ND 26 ND ND ND

16. Gu2 - 52 ND 42 52 24 51

17. Gu3 - 40 ND 55 40 24 55

18. Gu4 - 32 ND 43 32 34 ND

19. Gu5 ++ 54 ND 50 54 ND 67

20. Gy1 +++ 45 28 21 36 ND 30

21. Gy2 - 36 19 21 ND ND 22

22. N1 - 18 18 24 30 ND ND

23. N2 - 44 24 ND 45 ND ND

24. R1 +++ 18 ND 27 ND ND 34

25. R2 - 34 ND 38 30 ND 19

26. KV - 34 ND ND ND ND ND


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27. KC1 - 25 ND 29 37 24 ND

28. KC2 +++ 38 23 28 30 ND 39

29. KC3 - 31 60 35 60 57 ND

30. KC4 - 33 53 35 52 43 ND

31. KC5 +++ 19 18 23 ND ND 10

32. KC6 ++ 38 20 30 33 ND 27

33. KC7 ++ 49 ND 26 ND ND 26

34. KC8 ++ 38 21 39 52 45 ND

35. KB1 ++++ 44 40 31 ND ND 23

36. KB2 - 34 32 22 35 ND 31

37. KB3 - 40 32 21 ND ND 25

38. KB4 ++++ 34 ND 29 ND ND 40

39. M2 ++ 40 34 20 ND ND 27

40. M3 - 42 29 51 ND ND 25

41. M4 - 42 35 21 ND ND 32

42. MJ1 ++++ 28 ND 25 48 ND ND

43. MJ2 +++ 30 ND 34 35 ND 26

44. BII1 ++ 25 ND 30 38 ND ND

45. NPI1 ++ 25 ND 29 62 31 ND

46. NPI2 ++++ 28 26 35 ND ND 18

47. NPI4 - ND ND ND ND ND ND

48. NPI5 - 33 20 ND 19 ND 15

49. NPI6 - 41 29 ND 33 33 24

50. NPII1 +++ 35 ND ND 25 ND 32

51. NPII2 +++ 47 21 18 30 ND ND

52. NPII4 ++ 35 ND ND ND ND ND

53. NPII5 ++ 42 24 ND ND 23 ND

54. NPII6 - ND ND 50 ND ND ND

55. K - 44 ND ND ND 29 63


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[Note: Observations for GI production were noted by the presence of luxurious growth on the media and

denoted by (+) sign. All other enzyme productions are measured and listed as hydrolysis zone diameters

produced by the isolates].

Table 4.1a: Details of the symbols used for GI production in Table No. 4.1.

56. MR1 - 24 ND 18 36 ND ND

57. S1 - 33 24 ND ND ND ND

58. S2 ++ ND 26 ND ND ND 41

59. S4 - 42 44 41 ND ND

60. VJ1 ++++ 32 ND 30 ND ND ND

61. VJ2 +++ 38 ND ND ND ND ND

62. AI1 ++ 55 ND 40 ND ND 58

63. AI2 - ND 37 40 ND 44 ND

64. AII1 ++ 52 ND ND ND ND ND

65. AII2 ++ 37 47 48 47 ND 18

66. AII3 + 40 47 43 40 32 29

67. AII4 ++++ 25 ND 22 ND ND ND

68. AII5 ++++ 35 ND 37 61 51 ND

69. KNI1 - 45 ND 39 48 ND 27

70. KNI2 - 58 49 52 ND 27 65

71. KNII1 ++ 27 20 ND 45 ND ND

72. KNII2 ++ 40 ND 21 30 32 26

73. KNII3 - 50 ND ND ND ND ND

74. KNII4 ++ 44 ND 42 44 34 ND

75. LP - ND ND ND ND 28 ND

SYMBOL Response Inference

++++ Very Good Growth High GI production

+++ Good Growth Moderate GI production

++ Fair Growth Less GI production

+ Very Less Growth Negligible GI production

- No Growth No GI production

ND Not Detected No Enzyme Produced


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4.3.1.1 Glucose Isomerase Production

Each isolate responded differently on three media (X+P+ medium, X+P-

medium and wheat bran medium) combinations described in section 4.2.2.1.

Response of the isolates was best on wheat bran agar medium but their growth was

less flourished on X+P+ and X+P- media. The appearance of isolates started after 24

hours on the plates. The isolates giving early appearance on wheat bran also grew on

other two plates. X+P+ and X+P- media gave a clear picture of isolates as GI

producers, as former is containing xylose, peptone and mineral salts whereas latter

is containing only xylose and mineral salts. The growth of organism on X+P- media

(in absence of peptone) indicates the production of GI for utilizing xylose present in

the media. The isolates which grew well on both X+P+ and X+P- media were picked

up as good producers of GI and those growing only on X+P+ were grouped as

moderate GI producers. The isolates exhibited scanty growth on X+P- media. The

cultures selected by this method were further screened by subjecting to submerged

fermentation process [Manhas and Bala, 2004]. The results are depicted in Table No.

4.1 in the form of (+) sign. The number of (+) signs is directly proportional to the

growing capacity of the isolate on X+P- medium. A comparison of growth on all the

above media is shown in Fig. 4.1. The early appearance of GI producers is depicted in

Fig. 4.2.

Fig.4.1a

Fig.4.1a: Wheat bran agar plates showing the appearance ofout of 7 are exhibiting better growth after 48 h;scanty growth as compared to X+P+ medium;inoculated.

Fig.4.1b


a: Wheat bran agar plates showing the appearance of 7cultures out of 10 inoculated isolates. 3r growth after 48 h; b: X+P-medium c: X+P+ medium, X+P

scanty growth as compared to X+P+ medium; d: X+P- plates showing the growth of 4 isolates out of 10

Fig.4.1d

Fig.4.1b Fig.4.1c


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7cultures out of 10 inoculated isolates. 3: X+P+ medium, X+P- medium showing

plates showing the growth of 4 isolates out of 10

Fig.4.1c


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Fig. 4.2: Variation of growth observed after 24 and 48 hours, a: Appearance of P1 in 24 hours; b:

flourished growth of P1 on X+P- in 48 hours as compared to other isolates which are growing well

on X+P+ and wheat bran media.

Fig.4.2a

Fig.4.2b


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4.3.1.2 Amylase Production

Starch hydrolysis was observed as a common feature among the isolates

checked. Out of 75 isolates 40 were positive and 16 of these were prominently good

producers. The isolates KC3, KC4, V6, Ab, AII2 and AII3 were good amylase

producers. The isolate KC3 gave the highest zone diameter of 60 mm. The extensive

amylase production may be due to the adaptation of these isolates towards their

environmental conditions. The soil and compost pits are rich in plant and animal

wastes and starch is one of the most abundant polysaccharide components of plants.

In order to absorb nutrients from soil these microbes must be producing amylase

extracellularly to degrade starch and take up the solublised glucose. The plates

showing starch hydrolysis are shown in Fig. 4.3.

Fig. 4.3: Zone of starch hydrolysis produced by different isolates, a: P1; b: KC3; c: AII3; d: Ga3.

Fig.4.3a

Fig.4.3dFig.4.3c

Fig.4.3b

4.3.1.3 Cellulase production

Cellulase production was observed by the majority of the isolates. Nearly all

the isolates produced cellulase in more or less amounts.

produced by 70 out

V1, Ga1, KC7 and KNII3

The widespread capacity of producing cellulase by most of the isolates can be

accounted by the fact that major c

is easily available as a source of nutrient.

environment for their growth.

Fig. 4.4.

Fig. 4.4: Zone of cellulose hydrolysis produced by

Fig.4.4a

Fig.4.4c


Cellulase production


the isolates produced cellulase in more or less amounts. Measurable size of zone was

out of 75 isolates but 41 of them were good producers.

V1, Ga1, KC7 and KNII3 produced highest zones with 68 mm zone diameter for Ga1.


accounted by the fact that major component of plant waste in soil is cellulose, which

is easily available as a source of nutrient. The microbes adapt themselves to the

environment for their growth. The plates showing cellulose hydrolysis are shown in

of cellulose hydrolysis produced by some isolates, a: P1; b: Ga1; c: M4 d: AII3

Fig.4.4d

Fig.4.4b


126


Measurable size of zone was

of them were good producers. The isolates

mm zone diameter for Ga1.


omponent of plant waste in soil is cellulose, which

The microbes adapt themselves to the

The plates showing cellulose hydrolysis are shown in

some isolates, a: P1; b: Ga1; c: M4 d: AII3.

4.3.1.4 Protease production

The milk agar and casein agar plates started showing hydrolysis from the

second day. The zones increased on further incubation.

positive for gelatinase and

considerably large gelatin degradation zones

agar plates. A maximum of

gelatinase. The isolate

plate. The plates showing casein hydrolysis are shown in Fig. 4.

hydrolysis are shown in Fig. 4.

Fig. 4.5: Zone of casein hydrolysis produced by isolates

Fig. 4.6: Zone of gelatin hydrolysis produced by isolates

Fig.4.5a

Fig.4.6a


Protease production


second day. The zones increased on further incubation. Out of 75 cultures

elatinase and 41 for caseinase. Among these 9

considerably large gelatin degradation zones and 30 produced large zones on

maximum of 65 mm zone diameter was produced by

The isolate Ab also produced 65 mm zone diameter on casein containing

The plates showing casein hydrolysis are shown in Fig. 4.

hydrolysis are shown in Fig. 4.6.

: Zone of casein hydrolysis produced by isolates a: V2; b: V3; c: V6

: Zone of gelatin hydrolysis produced by isolates a: AII5; b: KC8; c:

Fig.4.6b

Fig.4.5cFig.4.5b


127


Out of 75 cultures 24 were

Among these 9 isolates exhibited

produced large zones on casein

was produced by Ab and Ga1 for

mm zone diameter on casein containing

The plates showing casein hydrolysis are shown in Fig. 4.5 and gelatin

; b: V3; c: V6.

a: AII5; b: KC8; c: Ab.

Fig.4.6c

Fig.4.5c


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4.3.1.5 Lipase production

The clear area around the colonies started developing after 48 h of incubation

which kept on increasing further. Many of these isolates are found to be lipolytic. In

all 58 out of 75 isolates gave clear zones on Tri-butyrene agar plates. Among these 25

were good producers with the maximum zone diameter of 55 mm for Gu3. M3, V5,

Ga4 and NPII6 also exhibited huge lipolytic zones.

4.3.1.6 Pectinase production

The pectinolytic activity was shown by 43 out of 75 isolates. Considerable

degradation capacity was exhibited by 12 of these isolates. The largest zone diameter

of 67 mm was produced by Gu5. The results are shown in Fig. 4.7.

Fig. 4.7: Zone of pectin hydrolysis produced by isolates a: Gu3; b: K and NPII1.

Fig.4.7a Fig.4.7b


4.3.2.1 Screening for Glucose

After checking the isolates by qualitative plate as

picked up for primary screening of GI production in submerged fermentation process.

Majority of the cultures exhibited the isomerisation capacity but 18 of these isolates

gave higher yields. The biomass started appearing in the

enzyme activity was higher for the isolates which appeared early on the xylose plates.

Many isolates were found to produce GI. The enzyme ranged from 0.97

2.8 Units/mL. Different isolates gave varying extent of growth whi

accounted by different medium requirements and growth phases of the isolates. The

medium used for primary screening did not contain any inducer as used by some

investigators [Bhosale et al

experiment was run in triplicates and the average of the activities obtained was used

to observe the results

The results for GI production in submerged fermentation are presented Fig.

Fig. 4.8: GI activity of different isolates.

0

0.5

1

1.5

2

2.5

3

3.5

P1 V3

Glu

cose

Isom

ers

eA

cti

vit

y[U

/m

l]



Screening for Glucose Isomerase production by submerged fermentation

After checking the isolates by qualitative plate assay method 36 cultures were



gave higher yields. The biomass started appearing in the


Many isolates were found to produce GI. The enzyme ranged from 0.97

2.8 Units/mL. Different isolates gave varying extent of growth whi



Bhosale et al., 1996; Hasal et al., 1992;

iment was run in triplicates and the average of the activities obtained was used

to observe the results. The highest producer pointed out in primary screening is P1.

The results for GI production in submerged fermentation are presented Fig.

: GI activity of different isolates.

V5 M2 KB1 KB4 Gy1 KC2 KC5 KC6 KC7 NPI2

Isolates


129

somerase production by submerged fermentation

say method 36 cultures were



gave higher yields. The biomass started appearing in the medium after 30 h. The


Many isolates were found to produce GI. The enzyme ranged from 0.97 Units/mL to

2.8 Units/mL. Different isolates gave varying extent of growth which can be



Chen et al., 1979]. The

iment was run in triplicates and the average of the activities obtained was used

The highest producer pointed out in primary screening is P1.

The results for GI production in submerged fermentation are presented Fig. 4.8.

NPI2 NPII1 AII4 MJ1 MJ2 VJ1 BII1

4.3.2.2 Screening for

More than 30 isolates exhibited observable hydrolytic zones on the plates. All of them

were subjected to submerged fermentation to check the enzyme

showed high activity ranging from

has been reported by

Bacillus sp. [Naidu et al.

production by submerged fermentation are presented in Fig. 4.

Fig. 4.9: Amylase activity of different isolates.

Kar et al., (2008

kiln soil. Extracellular

culture parameters for production

period (36 h). Soluble starch

and ammonium chloride

%) was most stimulatory in

al., (2009); Cotarlet et al.

researchers have also studied amylase production from

0

50

100

150

200

250

300

350

P1

V2

V3

V5

Am

yla

se

Acti

vit

y[U

/m

L]


Screening for Amylase production by submerged fermentation


were subjected to submerged fermentation to check the enzyme

showed high activity ranging from 9.28U/mL to 278.4 U/

has been reported by Streptomycetes [Kar et al., 2010; Yang

Naidu et al., 2009; Kurosawa et al., 2006].


: Amylase activity of different isolates.

2008) isolated Streptomyces erumpens MTCC 7317 from a brick

xtracellular amylase produced was moderately thermo

ulture parameters for production as pH (6.0), temperature (50°C) and incubation

oluble starch, beef extract, glycerol, yeast extract, peptone, casein

ammonium chloride promoted enzyme production. Glycerol concentrat

%) was most stimulatory in enzyme production. Manivasagan et al.

otarlet et al., (2009); Poornima et al., (2008

researchers have also studied amylase production from Streptomyces

V5

V6

V7

Ab

Ga3

Gy1

Gy2 R1

R2

M2

M3

KB

1

KB

3

KB

4

KC

1

KC

2

KC

3

KC

4

Isolates


130

production by submerged fermentation


were subjected to submerged fermentation to check the enzyme yield. Many of them

9.28U/mL to 278.4 U/mL. Amylase production

Yang and Wang, 1999 ] and

. The results for amylase

production by submerged fermentation are presented in Fig. 4.9.

MTCC 7317 from a brick-

moderately thermostable with optimum

pH (6.0), temperature (50°C) and incubation

yeast extract, peptone, casein

Glycerol concentration (0.02

Manivasagan et al., (2010), Kar et

2008) and various other

Streptomyces sp.

KC

4

KC

5

KC

6

KC

7

KC

8

NP

I2

NP

I5

KN

I1

AII

4

MJ2

VJ1


Cellulase production was determined in terms of filter paper activity

showed a wide range of activity from


screening for the possibility of good cellulase pr

reported to produce cellulase in large amount.

cloning of endoglucanase gene from a high cel

Fig. 4.10: Cellulase (FP

Chellapandi

sp. in solid state fermentation in 72

Streptomyces actuosus

the Vellar estuary exhibited

sp. have been immobilised in alginate beads by investigators for operation in stirred

tank reactor [Modi and Ray, 1996]

chloride promoted cellulase production at pH 7 and temperature 35°C.

al., (2010) studied

cellulase production in the presence of rice straw.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

P1

V1

V2

V3

V5

Fil

ter

Paper

Acti

vit

y[U

/m

L]


ning for Cellulase production by submerged fermentation

Cellulase production was determined in terms of filter paper activity

showed a wide range of activity from 0.06 to 1.3U/mL.


screening for the possibility of good cellulase producer, Streptomycetes have been

reported to produce cellulase in large amount. Cappa et al.

cloning of endoglucanase gene from a high cellulase producing

(FPAase) activity of different isolates.

Chellapandi and Jani, (2008) reported cellulase production by

sp. in solid state fermentation in 72-88 h. Murugan

treptomyces actuosus from the gut of estuarine finfish Mugil cephalus

exhibited cellulase activity. Cellulase produced by


[Modi and Ray, 1996]. Sucrose as a carbon source

chloride promoted cellulase production at pH 7 and temperature 35°C.

the optimisation of medium ingredients and

on in the presence of rice straw.

V5

V6

V7

Ab

Ga3

Gy1

Gy2

R1

R2

M2

M3

KB

1

KB

3

KB

4

KC

1

KC

2

KC

3

KC

4

KC

5

Isolates


131


Cellulase production was determined in terms of filter paper activity. The isolates

U/mL. The results for cellulase

are presented in Fig. 4.10. We at our end are

oducer, Streptomycetes have been

Cappa et al., (1997) has reported

lulase producing Streptomyces rochei.

reported cellulase production by Streptomyces

Murugan et al., (2007) isolated

Mugil cephalus collected from

Cellulase produced by Streptomyces


Sucrose as a carbon source and 1-2% sodium

chloride promoted cellulase production at pH 7 and temperature 35°C. El-Sersy et

the optimisation of medium ingredients and reported maximum

KC

5

KC

6

KC

7

KC

8

NPI2

NPI5

NPII

1

NPII

2

AII

4

AII

5

BII

1

MJ1

MJ2

VJ1


The cultures showed a wide range in protease production. There were good as

well as moderate producers of protease. The lowest productivity for an isolate

was determined as 1.98

is a good amylase as well as cellulase producer. This can be further picked up for

detailed studies. The results for protease production by submerged fermentation are

presented in Fig. 4.11

Fig. 4.11: Protease activity of different isolates.

Streptomycetes are

alkaline proteases, keratinases,

(2007) produced alkaline

clavuligerus.

De Azeredo

efficient degradation of chicken feather by

(2007) reported the

0

10

20

30

40

50

60

70

80

90

P1

V2

V3

V5

V7

Pro

tease

Acti

vit

y[U

/m

L]


Screening for Protease production by submerged fermentation


well as moderate producers of protease. The lowest productivity for an isolate

was determined as 1.98 U/mL and the highest was 76.56 U/mL for KC4. This isolate


The results for protease production by submerged fermentation are

11.

: Protease activity of different isolates.

Streptomycetes are well known producers of a wide variety of proteases like

alkaline proteases, keratinases, collagenase and pronase etc.

alkaline protease from a salt- tolerant and alkaliphilic

et al., (2006) and Bockle et al., (1995

efficient degradation of chicken feather by Streptomyces sp.

reported the production of collagenase enzyme from Thermoactinomyces

KB

1

KB

3

KB

4

V6

M2

M3

Ab

Ga3

R1

R2

Gy1

KC

1

KC

2

KC

3

KC

4

KC

5

KC

6

Isolates


132



well as moderate producers of protease. The lowest productivity for an isolate NPI5

U/mL for KC4. This isolate


The results for protease production by submerged fermentation are

well known producers of a wide variety of proteases like

and pronase etc. Thumar and Singh

tolerant and alkaliphilic Streptomyces

1995) demonstrated the

sp. Petrova and Vlahov,

Thermoactinomyces.

KC

7

KC

8

NPI2

NPI5

NPII

1

NPII

2

AII

4

AII

5

BII

1

MJ1

MJ2

VJ1


133

4.4 CONCLUSIONS

The detailed enzyme profiling of all the isolates indicate that they have

immense capacity of producing multiple extracellular enzymes in large amount. The

majority of samples were compost pits which contains huge amount of plant

remains. The excessive availability of cellulosic and starchy materials must have

enriched the number of cellulase and amylase producing organisms. The isolates

also exhibited good amount of pectinolytic activity. This enzyme is also helpful in

degrading large amount of pectin in plant material. Basic theme of our study is to

isolate a good glucose isomerase producer. An organism which produces amylase

and cellulase along with glucose isomerase will be of great commercial importance,

many of the GI producers detected here are also producing cellulase and amylase.

Some other isolates which are producing higher amounts of cellulase and amylase

can also be used for pretreatment of the starting material for HFCS production. An

advantageous result obtained at this level of investigation is that GI producing

isolates are also exhibiting amylase, cellulase and protease production which can

help in commercial applications. This will enable the growth of GI producers and

production of GI on economically available agro-residues. There are several reports

on using a cocktail of two organisms for performing simultaneous saccharification

and isomerisation. GI also finds application in ethanol production where it converts

non-fermentable sugars (xylose) to fermentable sugars (xylulose), combination of

saccharification, isomerisation can be helpful in ethanol fermentation at industrial

level.

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CHAPTER - IV - Shodhganga : a reservoir of Indian theses ...shodhganga.inflibnet.ac.in/bitstream/10603/4370/11/11...chocolate syrup, bread-making, fruit juices, paper and desizing